Enhanced Inflammasome Activation Research Articles

Formulating a TMEM176B blocker in chitosan nanoparticles uncouples its paradoxical roles in innate and adaptive antitumoral immunity

The immunoregulatory cation channel TMEM176B plays a dual role in tumor immunity. On the one hand, TMEM176B promotes antigen cross-presentation to CD8+ T cells by regulating phagosomal pH in dendritic cells (DCs). On the other hand, it inhibits NLRP3 inflammasome activation through ionic mechanisms in DCs, monocytes and macrophages. We speculated that formulating BayK8644 in PEGylated chitosan nanoparticles (NP-PEG-BayK8644) should slowly release the compound and by that mean avoid cross-presentation inhibition (which happens with a fast 30 min kinetics) while still triggering inflammasome activation.Chitosan nanocarriers were successfully obtained, exhibiting a particle size within the range of 200 nm; they had a high positive surface charge and a 99 % encapsulation efficiency. In in vitro studies, NP-PEG-BayK8644 did not inhibit antigen cross-presentation by DCs, unlike the free compound. The NP-PEG-BayK8644 activated the inflammasome in a Tmem176b-dependent manner in DCs. We administered either empty (eNP-PEG) or NP-PEG-BayK8644 to mice with established tumors. NP-PEG-BayK8644 significantly controlled tumor growth and improved mice survival compared to both eNP-PEG and free BayK8644 in melanoma and lymphoma models. This effect was associated with enhanced inflammasome activation by DCs in the tumor-draining lymph node and infiltration of the tumor by CD8+ T cells. Thus, encapsulation of BayK8644 in chitosan NPs improves the anti-tumoral properties of the compound by avoiding inhibition of antigen cross-presentation.

Diabetic Wound Keratinocytes Induce Macrophage JMJD3-Mediated Nlrp3 Expression via IL-1R Signaling.

Macrophage (Mφ) plasticity is critical for normal wound repair; however, in type 2 diabetic wounds, Mφs persist in a low-grade inflammatory state that prevents the resolution of wound inflammation. Increased NLRP3 inflammasome activity has been shown in diabetic wound Mφs; however, the molecular mechanisms regulating NLRP3 expression and activity are unclear. Here, we identified that diabetic wound keratinocytes induce Nlrp3 gene expression in wound Mφs through IL-1 receptor-mediated signaling, resulting in enhanced inflammasome activation in the presence of PAMPs and DAMPs. We found that IL-1 alpha is increased in human and murine wound diabetic keratinocytes compared to non-diabetic controls and directly induces Mφ Nlrp3 expression through IL-1 receptor signaling. Mechanistically, we report that the histone demethylase, JMJD3, is increased in wound Mφs late post-injury and is induced by IL-1 alpha from diabetic wound keratinocytes, resulting in Nlrp3 transcriptional activation through an H3K27me3-mediated mechanism. Using genetically engineered mice deficient in JMJD3 in myeloid cells (Jmjd3fl/fllyz2cre+), we demonstrate that JMJD3 controls Mφ-mediated Nlrp3 expression during diabetic wound healing. Thus, our data suggest a role for keratinocyte-mediated IL-1 alpha/IL-1R signaling in driving enhanced NLRP3 inflammasome activity in wound Mφs. These data also highlight the importance of cell crosstalk in wound tissues and identify JMJD3 and the ILR signaling cascade as important upstream therapeutic targets for Mφ NLRP3 inflammasome hyperactivity in nonhealing diabetic wounds.

ATG16L1 in myeloid cells limits colorectal tumor growth in ApcMin/+ mice infected with colibactin-producing Escherichia coli via decreasing inflammasome activation

ABSTRACT Escherichia coli strains producing the genotoxin colibactin, designated as CoPEC (colibactin-producing E. coli), have emerged as an important player in the etiology of colorectal cancer (CRC). Here, we investigated the role of macroautophagy/autophagy in myeloid cells, an important component of the tumor microenvironment, in the tumorigenesis of a susceptible mouse model infected with CoPEC. For that, a preclinical mouse model of CRC, the Apc Min/+ mice, with Atg16l1 deficiency specifically in myeloid cells (Apc Min/+ /Atg16l1[∆MC]) and the corresponding control mice (Apc Min/+ ), were infected with a clinical CoPEC strain 11G5 or its isogenic mutant 11G5∆clbQ that does not produce colibactin. We showed that myeloid cell-specific Atg16l1 deficiency led to an increase in the volume of colonic tumors in Apc Min/+ mice under infection with 11G5, but not with 11G5∆clbQ. This was accompanied by increased colonocyte proliferation, enhanced inflammasome activation and IL1B/IL-1β secretion, increased neutrophil number and decreased total T cell and cytotoxic CD8+ T cell numbers in the colonic mucosa and tumors. In bone marrow-derived macrophages (BMDMs), compared to uninfected and 11G5∆clbQ-infected conditions, 11G5 infection increased inflammasome activation and IL1B secretion, and this was further enhanced by autophagy deficiency. These data indicate that ATG16L1 in myeloid cells was necessary to inhibit colonic tumor growth in CoPEC-infected Apc Min/+ mice via inhibiting colibactin-induced inflammasome activation and modulating immune cell response in the tumor microenvironment. Abbreviation: AOM, azoxymethane; APC, APC regulator of WNT signaling pathway; ATG, autophagy related; Atg16l1[∆MC] mice, mice deficient for Atg16l1 specifically in myeloid cells; CASP1, caspase 1; BMDM, bone marrow-derived macrophage; CFU, colony-forming unit; CoPEC, colibactin-producing Escherichia coli; CRC, colorectal cancer; CXCL1/KC, C-X-C motif chemokine ligand 1; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MC, myeloid cell; MOI, multiplicity of infection; PBS, phosphate-buffered saline; pks, polyketide synthase; qRT-PCR, quantitative real-time reverse-transcription polymerase chain reaction; siRNA, small interfering RNA; TME, tumor microenvironment; TNF/TNF-α, tumor necrosis factor.

Transcription factor ASCL1 targets SLC6A13 to inhibit the progression of hepatocellular carcinoma via the glycine-inflammasome signaling.

The purpose of this research was to clarify the function of achaete-scute family bHLH transcription factor 1 (ASCL1) and solute carrier family 6 member 13 (SLC6A13) in influencing tumor cell behavior, inflammatory responses, and the regulation of inflammasomes. We analyzed the differentially expressed genes (DEGs) in the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database, as well as in the GSE14520 and GSE67764 datasets, to identify the expression changes of SLC6A13 in liver cancer. The prognostic significance of SLC6A13 in LIHC was assessed through Kaplan-Meier survival curve analysis. Transcriptional regulation of SLC6A13 by ASCL1 was explored using the Joint Annotation of the Human Genome and other species by the Systematic Pipeline for the Annotation of Regulatory Regions (JASPAR) database and dual-luciferase assays. In vitro experiments investigated the impact of ASCL1 and SLC6A13 overexpression on hepatocellular carcinoma (HCC) cell growth. Additionally, the effects of ethanol treatment and glycine modulation on the inflammatory response in HCC cell lines were evaluated. HCC samples showed reduced levels of SLC6A13, which correlates with a better prognosis for liver metastases. Elevated SLC6A13 expression correlated with improved overall survival (OS), progression-free survival (PFS), recurrence-free survival (RFS), and disease-specific survival (DSS). ASCL1 upregulated SLC6A13 and inhibited proliferation, migration, and invasion of HCC cells. Ethanol induced the production of inflammatory cytokines, which was enhanced by overexpression of SLC6A13 but counteracted by glycine. This study highlighted elevated expression of SLC6A13 in LIHC which has been correlated with improved OS, PFS, RFS, and DSS. Overexpression of SLC6A13 and ASCL1 in HCC cells enhanced inflammasome activation, which was exacerbated by ethanol and attenuated by glycine.

GITR exacerbates lysophosphatidylcholine-induced macrophage pyroptosis in sepsis via posttranslational regulation of NLRP3.

The NLRP3 inflammasome functions as an inflammatory driver, but its relationship with lipid metabolic changes in early sepsis remains unclear. Here, we found that GITR expression in monocytes/macrophages was induced by lysophosphatidylcholine (LPC) and was positively correlated with the severity of sepsis. GITR is a costimulatory molecule that is mainly expressed on T cells, but its function in macrophages is largely unknown. Our in vitro data showed that GITR enhanced LPC uptake by macrophages and specifically enhanced NLRP3 inflammasome-mediated macrophage pyroptosis. Furthermore, in vivo studies using either cecal ligation and puncture (CLP) or LPS-induced sepsis models demonstrated that LPC exacerbated sepsis severity/lethality, while conditional knockout of GITR in myeloid cells or NLRP3/caspase-1/IL-1β deficiency attenuated sepsis severity/lethality. Mechanistically, GITR specifically enhanced inflammasome activation by regulating the posttranslational modification (PTM) of NLRP3. GITR competes with NLRP3 for binding to the E3 ligase MARCH7 and recruits MARCH7 to induce deacetylase SIRT2 degradation, leading to decreasing ubiquitination but increasing acetylation of NLRP3. Overall, these findings revealed a novel role of macrophage-derived GITR in regulating the PTM of NLRP3 and systemic inflammatory injury, suggesting that GITR may be a potential therapeutic target for sepsis and other inflammatory diseases. GITR exacerbates LPC-induced macrophage pyroptosis in sepsis via posttranslational regulation of NLRP3. According to the model, LPC levels increase during the early stage of sepsis, inducing GITR expression on macrophages. GITR not only competes with NLRP3 for binding to the E3 ligase MARCH7 but also recruits MARCH7 to induce the degradation of the deacetylase SIRT2, leading to decreasing ubiquitination but increasing acetylation of NLRP3 and therefore exacerbating LPC-induced NLRP3 inflammasome activation, macrophage pyroptosis and systemic inflammatory injury.

Cytotoxic Lymphocyte-Monocyte Complex Reflects the Dynamics of Coronavirus Disease 2019 Systemic Immune Response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes a variety of clinical manifestations, many of which originate from altered immune responses, either locally or systemically. Immune cell cross-talk occurs mainly in lymphoid organs. However, systemic cell interaction specific to coronavirus disease 2019 has not been well characterized. Here, by employing single-cell RNA sequencing and imaging flow cytometry analysis, we unraveled, in peripheral blood, a heterogeneous group of cell complexes formed by the adherence of CD14+ monocytes to different cytotoxic lymphocytes, including SARS-CoV-2-specific CD8+ T cells, γδ T cells, and natural killer T cells. These lymphocytes attached to CD14+ monocytes that showed enhanced inflammasome activation and pyroptosis-induced cell death in progression stage; in contrast, in the convalescent phase, CD14+ monocytes with elevated antigen presentation potential were targeted by cytotoxic lymphocytes, thereby restricting the excessive immune activation. Collectively, our study reports previously unrecognized cell-cell interplay in the SARS-CoV-2-specific immune response, providing new insight into the intricacy of dynamic immune cell interaction representing antiviral defense.

Downregulation of the kidney glucagon receptor, essential for renal function and systemic homeostasis, contributes to chronic kidney disease

The glucagon receptor (GCGR) in the kidney is expressed in nephron tubules. In humans and animal models with chronic kidney disease, renal GCGR expression is reduced. However, the role of kidney GCGR in normal renal function and in disease development has not been addressed. Here, we examined its role by analyzing mice with constitutive or conditional kidney-specific loss of the Gcgr. Adult renal Gcgr knockout mice exhibit metabolic dysregulation and a functional impairment of the kidneys. These mice exhibit hyperaminoacidemia associated with reduced kidney glucose output, oxidative stress, enhanced inflammasome activity, and excess lipid accumulation in the kidney. Upon a lipid challenge, they display maladaptive responses with acute hypertriglyceridemia and chronic proinflammatory and profibrotic activation. In aged mice, kidney Gcgr ablation elicits widespread renal deposition of collagen and fibronectin, indicative of fibrosis. Taken together, our findings demonstrate an essential role of the renal GCGR in normal kidney metabolic and homeostatic functions. Importantly, mice deficient for kidney Gcgr recapitulate some of the key pathophysiological features of chronic kidney disease.

Distinct gene expression signatures comparing latent tuberculosis infection with different routes of Bacillus Calmette-Guérin vaccination

Tuberculosis remains an international health threat partly because of limited protection from pulmonary tuberculosis provided by standard intradermal vaccination with Bacillus of Calmette and Guérin (BCG); this may reflect the inability of intradermal vaccination to optimally induce pulmonary immunity. In contrast, respiratory Mycobacterium tuberculosis infection usually results in the immune-mediated bacillary containment of latent tuberculosis infection (LTBI). Here we present RNA-Seq-based assessments of systemic and pulmonary immune cells from LTBI participants and recipients of intradermal and oral BCG. LTBI individuals uniquely display ongoing immune activation and robust CD4 T cell recall responses in blood and lung. Intradermal BCG is associated with robust systemic immunity but only limited pulmonary immunity. Conversely, oral BCG induces limited systemic immunity but distinct pulmonary responses including enhanced inflammasome activation potentially associated with mucosal-associated invariant T cells. Further, IL-9 is identified as a component of systemic immunity in LTBI and intradermal BCG, and pulmonary immunity following oral BCG.

Enhanced inflammasome-mediated inflammation and impaired autophagy in peripheral blood mononuclear cells is associated with non-alcoholic fatty liver disease severity

AimsIdentification of the progress of non-alcoholic fatty liver disease (NAFLD) is crucial for their effective treatment. Circulating peripheral blood mononuclear cells (PBMC) could be a surrogate monitor instead of complicated and expensive biopsies. Changes in immuno-metabolic status in NAFLD patients may be reflected by an expression of different PBMC-specific molecular markers. It was hypothesized that impaired autophagy with enhanced inflammasome activation is a critical molecular event in PBMC that could contribute to systemic inflammation associated with NAFLD progression. Main methodsA cross-sectional study with a sample size of 50 subjects were undertaken from a governmental facility in Kolkata, India. Major anthropometric, biochemical, and dietary parameters were recorded. Cellular and serum samples of NAFLD patients were analyzed for oxidative stress, inflammation, inflammasome activation, and autophagic flux by western blot, flow cytometry, immunocytochemistry. Key findingsBaseline anthropometric and clinical parameters were found associated with NAFLD severity. Elevated systemic inflammation was reflected by higher proinflammatory markers like iNOS, Cox-2, IL-6, TNF-α, IL-1β, hsCRP in the serum of NAFLD subjects (p < 0.05). ROS-induced NLRP3 inflammasomes marker proteins were upregulated (p < 0.05) in PBMC along with NAFLD severity. Expression of autophagic markers such as LC3B, Beclin-1 and its regulator pAMPKα were found diminished (p < 0.05) with a concomitant rise of p62. Colocalization of NLRP3 with LC3B proteins in PBMC was found diminished along NAFLD severity. SignificancePresent data provide mechanistic evidence of impaired autophagy and intracellular ROS triggered inflammasome activation in PBMC, which could potentially exacerbate NAFLD severity.

Heterozygosity for Crohn’s Disease Risk Allele of Atg16L1 Protects from Salmonella Infection

Abstract Autophagy is an evolutionarily conserved pathway for the formation and trafficking of lipid vesicles in response to stress, including inflammation and microbial infection. A missense variant of the essential autophagy gene Atg16L1 (rs2241880, T300A) is carried by a majority of the human population and is linked to Crohn’s Disease (CD). Previous studies found that T300A enhances proteolytic cleavage and degradation of Atg16L1, leading to dysregulated cytokine expression and impaired control of microbial infection in knock-in mice. However, these studies have focused on homozygous animals while the plurality of the human population is heterozygous for the allele. Here we demonstrate that heterozygosity for T300A protects mice from Salmonella infection. We find the variant allele results in altered complex formation independent of cleavage, allowing for enhanced inflammasome activation but impaired conjugation of Atg8 to single membranes (CASM). In vivo, one copy of T300A is sufficient to elevate the production of inflammatory cytokines, which is required for protection from Salmonella infection. Meanwhile, mice deficient in CASM genes are susceptible to Salmonella infection and this pathway is shown to be impaired in homozygous but not heterozygous mice. Finally, we demonstrate that heterozygosity in human macrophages also results in elevated cytokine expression, while maintaining functional non-canonical autophagy. These results clarify the altered autophagy function of the CD risk allele Atg16L1T300A and suggest it can be beneficial to heterozygous carriers, providing a potential explanation for its prevalence in the human population. NIH R01 AI121244 F31 HL149238

Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

Ménière’s disease, a multifactorial disorder of the inner ear, is characterized by severe vertigo episodes and hearing loss. Although the role of immune responses in Ménière’s disease has been proposed, the precise mechanisms remain undefined. Here, we show that downregulation of serum/glucocorticoid-inducible kinase 1 is associated with activation of NLRP3 inflammasome in vestibular-resident macrophage-like cells from Ménière’s disease patients. Serum/glucocorticoid-inducible kinase 1 depletion markedly enhances IL-1β production which leads to the damage of inner ear hair cells and vestibular nerve. Mechanistically, serum/glucocorticoid-inducible kinase 1 binds to the PYD domain of NLRP3 and phosphorylates it at Serine 5, thereby interfering inflammasome assembly. Sgk−/− mice show aggravated audiovestibular symptoms and enhanced inflammasome activation in lipopolysaccharide-induced endolymphatic hydrops model, which is ameliorated by blocking NLRP3. Pharmacological inhibition of serum/glucocorticoid-inducible kinase 1 increases the disease severity in vivo. Our studies demonstrate that serum/glucocorticoid-inducible kinase 1 functions as a physiologic inhibitor of NLRP3 inflammasome activation and maintains inner ear immune homeostasis, reciprocally participating in models of Ménière’s disease pathogenesis.

Disruptions in endocytic traffic contribute to the activation of the NLRP3 inflammasome.

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and is regulated by subcellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endolysosomal markers and for the inositol lipid PI4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod, driving enhanced inflammasome activation and cytokine secretion. Together, these data suggest that NLRP3 can sense disruptions in the trafficking of endosomal cargoes, which may explain in part the spatial activation of the NLRP3 inflammasome. These data highlight mechanisms that could be exploited in the therapeutic targeting of NLRP3.

Intravesical BCG in patients with non-muscle invasive bladder cancer induces trained immunity and decreases respiratory infections

BackgroundBCG is recommended as intravesical immunotherapy to reduce the risk of tumor recurrence in patients with non-muscle invasive bladder cancer (NMIBC). Currently, it is unknown whether intravesical BCG application induces...

CGAS deficiency enhances inflammasome activation in macrophages and inflammatory pathology in pristane-induced lupus.

Type I interferon (IFN) plays a vital role in the pathogenesis of systemic lupus erythematosus. Cyclic GMP AMP synthase (cGAS) is a cytosolic DNA sensor that recognizes dsDNA and creates cGAMP to activate STING-mediated type I IFN production. The activation of STING induces lupus disease in Fcgr2b deficient mice through the differentiation of dendritic cells. In contrast, Cgas-deficient mice could be generated more autoantibody production and proteinuria in pristane-induced lupus (PIL). These data suggested that the other dsDNA sensors could be involved in lupus development mechanisms. This study aimed to identify the cGAS-mediated mechanisms contributing to lupus pathogenesis in PIL. The Cgas-deficient and WT mice were induced lupus disease with pristane and subsequently analyzed autoantibody, histopathology, and immunophenotypes. The lung tissues were analyzed with the expression profiles by RT-PCR and western blot. The bone marrow-derived macrophages were stimulated with inflammasome activators and observed pyroptosis. The Cgas-/- mice developed more severe pulmonary hemorrhage and autoantibody production than WT mice. The activated dendritic cells, IFN-g-, and IL-17a-producing T helper cells, and infiltrated macrophages in the lung were detected in Cgas-/- mice higher than in WT mice. We observed an increase in expression of Aim2, Casp11, and Ifi16 in the lung and serum IL-1a but IL-1b in pristane-injected Cgas-/- mice. The rise of Caspase-11 in the lung of pristane-injected Cgas-/- mice suggested noncanonical inflammasome activation. The activation of AIM2 and NLRP3 inflammasomes in bone marrow-derived macrophages (BMDMs) enhanced the number of dead cells in Cgas-/- mice compared with WT mice. Activation of the inflammasome significantly induced pyroptosis in Cgas-/- BMDMs. The dsDNA level, but not mitochondrial DNA, increased dramatically in pristane-injected Cgas-/- mice suggesting the dsDNA could be a ligand activating inflammasomes. The cGAS agonist-induced BMDM activation in the Cgas-/- mice indicated that the activation of DNA sensors other than cGAS enhanced activated macrophages. These findings suggested that cGAS hampers the unusual noncanonical inflammasome activation through other DNA sensors.

Interleukin-1 and the NLRP3 inflammasome in COVID-19: Pathogenetic and therapeutic implications.

SummaryA hyperinflammatory response during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection crucially worsens clinical evolution of coronavirus disease 2019 (COVID-19). The interaction between SARS-CoV-2 and angiotensin-converting enzyme 2 (ACE2) triggers the activation of the NACHT, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome. Enhanced inflammasome activity has been associated with increased disease severity and poor prognosis. Evidence suggests that inflammasome activation and interleukin-1β (IL-1β) release aggravate pulmonary injury and induce hypercoagulability, favoring progression to respiratory failure and widespread thrombosis eventually leading to multiorgan failure and death. Observational studies with the IL-1 blockers anakinra and canakinumab provided promising results. In the SAVE-MORE trial, early treatment with anakinra significantly shortened hospital stay and improved survival in patients with moderate-to-severe COVID-19. In this review, we summarize current evidence supporting the pathogenetic role of the NLRP3 inflammasome and IL-1β in COVID-19, and discuss clinical trials testing IL-1 inhibition in COVID-19.

Irisin inhibits NLRP3 inflammasome activation in HG/HF incubated cardiac microvascular endothelial cells with H/R injury.

NLRP3 inflammasome mediates myocardial ischemia/reperfusion (MI/R) injury and diabetic vascular endothelia dysfunction. However, the role of NLRP3 inflammasome in MI/R injury with diabetes has not been fully described. Irisin plays an important role in anti-inflammation and improves endothelial function in type 2 diabetes. The current study aimed to investigate the effect of irisin on regulating NLRP3 inflammasome activation in diabetic vascular endothelia dysfunction. Cardiac microvascular endothelial cells (CMECs) were cultured and subjected to high glucose/high fat (HG/HF) receiving hypoxia/reoxygenation (H/R) with irisin incubation or not. Then, apoptosis, viability, migration, NO secretion, and inflammasome activation were examined. The hypoxic CMECs exhibited increased apoptosis, impaired viability, and migration, even decreased NO secretion and enhanced inflammasome activation. Moreover, irisin incubation decreased NLRP3 activation and attenuated cell injury in HG/HF cultured CMECs subjected to H/R injury, which was abolished by NLRP3 inflammasome activation. Meanwhile, NLRP3 inflammasome siRNA also attenuated H/R injury in CMECs under HG/HF condition. The current study demonstrated for the first time that irisin inhibits NLRP3 inflammasome activation in CMECs as a novel mechanism in myocardial ischemia/reperfusion injury in diabetes.

A Randomized Open-Label Clinical Trial of Lipid-Lowering Therapy in Psoriasis to Reduce Vascular Endothelial Inflammation

In psoriasis, immune systemic activation and cardiometabolic abnormalities, including dyslipidemia, play a role in promoting impaired endothelial health and increased cardiovascular (CV) risk ( Greb et al., 2016 Greb J.E. Goldminz A.M. Elder J.T. Lebwohl M.G. Gladman D.D. Wu J.J. et al. Psoriasis. Nat Rev Dis Primers. 2016; 2: 16082 Crossref PubMed Scopus (427) Google Scholar ). Patients with psoriasis have elevated brachial vein endothelial proinflammatory transcript expression (impaired vascular endothelial health), with the degree of impairment directly correlated with skin activity and circulating biomarkers of systemic inflammation ( Garshick et al., 2019 Garshick M.S. Barrett T.J. Wechter T. Azarchi S. Scher J.U. Neimann A. et al. Inflammasome signaling and impaired vascular health in psoriasis. Arterioscler Thromb Vasc Biol. 2019; 39: 787-798 Crossref PubMed Scopus (34) Google Scholar ). In psoriasis, TNF and IL-17A synergism promote inflammasome (IL-1β) signaling and downstream IL-6 production (driver of CRP) ( Verma et al., 2021 Verma D. Fekri S.Z. Sigurdardottir G. Bivik Eding C. Sandin C. Enerbäck C. Enhanced inflammasome activity in patients with psoriasis promotes systemic inflammation. J Invest Dermatol. 2021; 141: 586-595.e5 Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar ), which associates with vascular endothelial inflammatory transcript expression, suggesting a pathogenic (immune-mediated) mechanism of atherosclerosis development ( Garshick et al., 2019 Garshick M.S. Barrett T.J. Wechter T. Azarchi S. Scher J.U. Neimann A. et al. Inflammasome signaling and impaired vascular health in psoriasis. Arterioscler Thromb Vasc Biol. 2019; 39: 787-798 Crossref PubMed Scopus (34) Google Scholar ). Is It Prime Time for Statin Therapy in Psoriasis?Journal of Investigative DermatologyVol. 142Issue 6PreviewIt has been over a decade and a half since the first large-scale study of the accelerated risk of myocardial infarction in psoriasis (Gelfand et al., 2006). Since then, a broad range of scientific approaches has determined that psoriasis itself is associated with a higher-risk subclinical vascular disease (Naik et al., 2015), high-risk coronary plaque (Lerman et al., 2017), and major adverse cardiovascular (CV) events (Mehta et al., 2010) and mortality (Noe et al., 2018). Although these studies have established that the risk of CV disease (CVD) is independent of traditional CV risk factors, psoriasis also has complex interactions with metabolic disease. Full-Text PDF

Abstract 14023: PRG4 in Calcified Atherosclerotic Plaque Induces Immune Tolerance in Macrophages, and is Implicated in Inflammation-Driven Vascular Calcification

Atherosclerotic calcification is an independent predictor for a cardiovascular event, and its development is driven by macrophage-led inflammation. PRG4 was previously shown to be the most upregulated gene in microarray data of carotid endarterectomy-derived, high vs low-calcified atherosclerotic plaques. It was shown to be a pro-calcifying factor in the disease, modulating the pro-calcifying switch of vascular smooth muscle cells (VSMC). PRG4’s role in calcified plaque is largely unknown, however, we are able to show a profound effect on macrophage immune response. Transcriptomic data of a highly calcified plaque cohort (N=18 patients) shows PRG4 expression is correlated with inflammatory gene pathways, including toll-like receptor signalling; and immunohistochemistry of this cohort displays co-localization with CD68 + cells. Despite this, PRG4 is not expressed in macrophages in plaque single-cell datasets or transcriptomics datasets, and plaque origin is likely fibroblast and VSMC. Human peripheral blood mononuclear cell (PBMC)-derived macrophage incubation with 100μg/ml recombinant human (rh)PRG4 shows strong NF-κB activation, and secretion of TNF, IL-6, IL-12p70 and IL-10. rhPRG4 stimulation also significantly enhances inflammasome activation, increasing ASC activation (p&lt;0.003) and IL-1β secretion (p&lt;0.001). Moreover, it suppressed macrophage phagocytosis of both zymosan particles (p&gt;0.001), and hydroxyapatite particles (p=0.004). Subsequently, cells stimulated with rhPRG4 develop immune tolerance, a suppressed inflammatory response, to secondary stimulation with either rhPRG4 or lipopolysaccharide - with almost complete ablation of TNF secretion (p&lt;0.001). This phenomenon is accompanied by a 5-fold A20 upregulation (p&lt;0.001), a well-established key regulator of endotoxin tolerance. This immunosuppressive effect was coupled with increased resistance to mitochondrial stress (p&lt;0.001). In conclusion, we see a PRG4-specific phenotype in macrophages; suppressed phagocytosis capability, and acute pro-inflammatory activation of NF-κB and inflammasome pathways, that over longer exposure develops into immunosuppression. This may have profound implications for the overall disease mechanism.

Dynamin-related protein 1 deficiency accelerates lipopolysaccharide-induced acute liver injury and inflammation in mice

Mitochondrial fusion and fission, which are strongly related to normal mitochondrial function, are referred to as mitochondrial dynamics. Mitochondrial fusion defects in the liver cause a non-alcoholic steatohepatitis-like phenotype and liver cancer. However, whether mitochondrial fission defect directly impair liver function and stimulate liver disease progression, too, is unclear. Dynamin-related protein 1 (DRP1) is a key factor controlling mitochondrial fission. We hypothesized that DRP1 defects are a causal factor directly involved in liver disease development and stimulate liver disease progression. Drp1 defects directly promoted endoplasmic reticulum (ER) stress, hepatocyte death, and subsequently induced infiltration of inflammatory macrophages. Drp1 deletion increased the expression of numerous genes involved in the immune response and DNA damage in Drp1LiKO mouse primary hepatocytes. We administered lipopolysaccharide (LPS) to liver-specific Drp1-knockout (Drp1LiKO) mice and observed an increased inflammatory cytokine expression in the liver and serum caused by exaggerated ER stress and enhanced inflammasome activation. This study indicates that Drp1 defect-induced mitochondrial dynamics dysfunction directly regulates the fate and function of hepatocytes and enhances LPS-induced acute liver injury in vivo.

Hypoxia and Porphyromonas gingivalis-lipopolysaccharide synergistically induce NLRP3 inflammasome activation in human gingival fibroblasts

ObjectiveTo investigate the effects of hypoxia and Porphyromonas gingivalis- lipopolysaccharide (P. gingivalis-LPS) on activation of the NACHT leucine-rich repeat protein 3 (NLRP3) inflammasome in human gingival fibroblasts (HGFs). DesignPeriodontitis was optimally simulated using a hypoxic concentration of 1%. HGFs were stimulated using P. gingivalis-LPS (1.0 μg/ml) in normoxia and hypoxia for 3 h and 6 h, respectively. The expression levels of genes and proteins of hypoxia-inducible factor-1α (HIF-1α), interleukin-1β, gasdermin D (GSDMD) and the NLRP3 inflammasome, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1 and its activated forms, were measured using quantitative real-time polymerase chain reaction and western blot. ELISA was used to detect and determine levels of the inflammatory factor interleukin-1β in cell supernatants. Lactate dehydrogenase (LDH) release assay, caspase-1 activity assay and Hoechst 33342/Propidium Iodide (PI) staining were performed to further verify the presence of pyroptosis. ResultsThe NLRP3 inflammasome (i.e., NLRP3, ASC, caspase-1) was not affected by individual stimulation using P. gingivalis-LPS or hypoxia. However, the combination of both hypoxia and P. gingivalis-LPS stimulation significantly enhanced inflammasome activation and promoted the expression of interleukin-1β, gasdermin D and HIF-1α at gene and protein levels; PI positive cells and the release of LDH were also elevated. ConclusionHypoxia and P. gingivalis-LPS synergistically induced NLRP3 inflammasome activation in HGFs, and subsequently high levels of interleukin-1β and GSDMD-mediated pyroptosis can cause an HGF inflammatory response, which plays an important role in the pathogenesis of periodontitis.